The present invention relates to the fields of medicine and molecular biology. More specifically, the invention provides nucleic acid molecules and proteins encoded thereby which are involved in the development of resistance to pharmacological and chemotherapeutic agents in tumor cells.
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
P-glycoprotein, the product of the MDR1 gene, was the first ABC transporter shown to confer resistance to cytotoxic agents. Pgp functions as an ATP-dependent efflux pump that reduces the intracellular concentration of a variety of chemotherapeutic agents by transporting them across the plasma membrane (1). The multidrug resistance phenotype associated with overexpression of Pgp is of considerable clinical interest because natural product drugs are second only to alkylating agents in clinical utility, and many effective chemotherapeutic regimens contain more than one natural product agent. More recently, we and others have reported transfection studies indicating that MRP, another ABC family transporter, confers a multidrug resistance phenotype that includes many natural product drugs, but is distinct from the resistance phenotype associated with Pgp (2-6). MRP shares only limited amino acid identity with Pgp, and this is reflected in the different substrate specificities of the two transporters. In contrast to Pgp, MRP can transport a wide range of anionic organic conjugates, including glutathione S-conjugates (7). In addition to Pgp and MRP there may be other transporters that are involved in cytotoxic drug resistance. In the case of natural product drugs, resistant cell lines have been described that display a multidrug resistant phenotype associated with a drug accumulation deficit, but do not overexpress Pgp or MRP (8). ABC transporters have also been linked to cisplatin resistance, and several lines of evidence suggest the possibility that pumps specific for organic anions may be involved: 1) decreased cisplatin accumulation is consistently observed in cisplatin resistant cell lines (9); 2) cisplatin is conjugated to glutathione in the cell, and this anionic conjugate is toxic in an in vitro biochemical assay (10); and 3) biochemical studies using membrane vesicle preparations have shown that cisplatin resistant cells lines have enhanced expression of an ATP-dependent transporter of CDDP-glutathione and other glutathione S-conjugates such as the cystinyl leukotriene LTC, (11, 12). These data thus suggest that an organic anion transporter may contribute to cisplatin resistance by exporting CDDP-glutathione. While MRP is an organic anion transporter, the reported drug resistance profile of MRP-transfected cells does not extend to this agent (5, 6), and to date only one cisplatin-resistant cell line has been reported to overexpress MRP (13). This suggests that organic anion transporters other than MRP may contribute to cisplatin resistance. Consistent with this possibility, the canalicular multispecific organic anion transporter, cMOAT, an MRP-related transporter that functions as the major organic anion transporter in liver, has been reported to be overexpressed in cisplatin resistant cell lines (14, 15). A more direct link between cMOAT and cytotoxic drug resistance is suggested by a recent report in which transfection of a cMOAT antisense construct into a liver cancer cell line resulted in sensitization to cisplatin, daunorubicin and other cytotoxic agents (16).
Clearly, a need exists for identifying the essential components and mechanisms giving rise to drug resistance and the transport of anticancer agents out of the tumor cell. The elucidation of these mechanisms may be used to advantage for the design of efficacious chemotherapeutic agents.
This invention provides novel, biological molecules useful for identification, detection, and/or molecular characterization of components involved in the acquisition of drug resistance in tumor cells. According to one aspect of the invention, an isolated nucleic acid molecule is provided which includes a sequence encoding a protein transporter of a size between about 1300 and 1350 amino acids in length. The encoded protein, referred to herein as MOAT-B, comprises a multi-domain structure including a tandem repeat of nucleotide binding folds appended C-terminal to a hydrophobic domain that contains several potential membrane spanning helices. Conserved Walker A and B ATP binding sites are present in each of the nucleotide binding folds.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human MOAT-B protein. In a particularly preferred embodiment, the human MOAT-B protein has an amino acid sequence the same as Sequence I.D. No. 2. An exemplary MOAT-B nucleic acid molecule of the invention comprises Sequence I.D. No. 1.
According to another aspect of the invention, a second isolated nucleic acid molecule is provided which includes a sequence encoding a transporter between about 1400 and 1450 amino acids. The encoded protein, referred to herein as MOAT-C contains a multi-domain structure including a tandem repeat of nucleotide binding folds appended C-terminal to a hydrophobic domain that contains several potential membrane spanning helices. Conserved Walker A and B ATP binding sites are present in each of the nucleotide binding folds. While similar in structure to MOAT-B described above, MOAT-C contains distinct sequence differences.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a human MOAT-C protein. In a particularly preferred embodiment, the human MOAT-C protein has an amino acid sequence the same as Sequence I.D. No. 4. An exemplary MOAT-C nucleic acid molecule of the invention comprises Sequence I.D. No. 3.
According to yet another aspect of the invention, an isolated nucleic acid molecule is provided which includes a sequence encoding a protein of a size between about 1500 and 1550 amino acids in length. The encoded protein, referred to herein as MOAT-D, contains a multidomain structure including an N-terminal hydrophobic extension which harbors five transmembrane spanning helices.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a MOAT-D protein. In a particularly preferred embodiment, the human MOAT-D protein has an amino acid sequence the same as Sequence I.D. No. 6. An exemplary MOAT-D nucleic acid molecule of the invention comprises Sequence I.D. No. 5.
According to yet another aspect of the invention, an isolated nucleic acid molecule is provided which includes a sequence encoding a protein of a size between about 1480 and 1530 amino acids in length. The encoded protein, referred to herein as MOAT-E, contains a multidomain structure including an N-terminal hydrophobic extension which harbors several transmembrane spanning helices. While similar in structure to MOAT-D described above, MOAT-E contains distinct sequence differences.
In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that includes a cDNA encoding a MOAT-E protein. In a particularly preferred embodiment, the human MOAT-E protein has an amino acid sequence the same as Sequence I.D. No. 8. An exemplary MOAT-E nucleic acid molecule of the invention comprises Sequence I.D. No. 7.
According to another aspect of the present invention, an isolated nucleic acid molecule is provided, which has a sequence selected from the group consisting of: (1) Sequence I.D. No. 1; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 1 comprising nucleic acids encoding amino acids 1-1154 of Sequence ID No. 2; (3) a sequence encoding preselected portions of Sequence I.D. No. 1 within nucleotides 1-3462, (4) Sequence I.D. No. 3; (5) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 3 comprising nucleic acids encoding amino acids 1-442 of Sequence ID No. 4; (6) a sequence encoding preselected portions of Sequence I.D. No. 3 within nucleotides 1-1326, (7) Sequence I.D. No. 5; (8) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 5 comprising nucleic acids encoding amino acids 1-1036 of Sequence ID No. 6; (9) a sequence encoding preselected portions of Sequence I.D. No. 5 within nucleotides 1-3108, (1) Sequence I.D. No. 7; (2) a sequence specifically hybridizing with preselected portions or all of the complementary strand of Sequence I.D. No. 7 comprising nucleic acids encoding amino acids 1-998 of Sequence ID No. 8; (3) a sequence encoding preselected portions of Sequence I.D. No. 7 within nucleotides 1-300.
Such partial sequences are useful as probes to identify and isolate homologues of the MOAT genes of the invention. Additionally, isolated nucleic acid sequences encoding natural allelic variants of the nucleic acids of Sequence I.D. Nos., 1, 3, 5 and 7 are also contemplated to be within the scope of the present invention. The term natural allelic variants will be defined hereinbelow.
According to another aspect of the present invention, antibodies immunologically specific for the human MOAT proteins described hereinabove are provided.
In yet another aspect of the invention, host cells comprising at least one of the MOAT encoding nucleic acids are provided. Such host cells include but are not limited to bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells. Host cells overexpressing one or more of the MOAT encoding nucleic acids of the invention provide valuable research tools for assessing transport of chemotherapeutic agents out of cells. MOAT expressing cells also comprise a biological system useful in methods for identifying inhibitors of the MOAT transporters.
Another embodiment of the present invention encompasses methods for screening cells expressing MOAT encoding nucleic acids for chemotherapy resistance. Such methods will provide the clinician with data which correlates expression of a particular MOAT genes with a particular chemotherapy resistant phenotype.
Diagnostic methods are also contemplated in the present invention. Accordingly, suitable oligonucleotide probes are provided which hybridize to the nucleic acids of the invention. Such probes may be used to advantage in screening biopsy samples for the expression of particular MOAT genes. Once a tumor sample has been characterized as to the MOAT gene(s) expressed therein, inhibitors identified in the cell line screening methods described above may be administered to prevent efflux of the beneficial chemotherapeutic agents from cancer cells.
The methods of the invention may be applied to kits. An exemplary kit of the invention comprises MOAT gene specific oligonucleotide probes and/or primers, MOAT encoding DNA molecules for use as a positive control, buffers, and an instruction sheet. A kit for practicing the cell line screening method includes frozen cells comprising the MOAT genes of the invention, suitable culture media, buffers and an instruction sheet.
In a further aspect of the invention, transgenic knockout mice are disclosed. Mice will be generated in which at least one MOAT gene has been knocked out. Such mice will provide a valuable in biological system for assessing resistance to chemotherapy in an in vivo tumor model.
Various terms relating to the biological molecules of the present invention are used hereinabove and also throughout the specification and claims. The terms xe2x80x9cpercent similarityxe2x80x9d and xe2x80x9cpercent identity (identical)xe2x80x9d are used as set forth in the UW GCG Sequence Analysis program (Devereux et al. NAR 12:387-397 (1984)).
With reference to nucleic acids of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5xe2x80x2 and 3xe2x80x2 directions) in the naturally occurring genome of the organism from which it originates. For example, the xe2x80x9cisolated nucleic acidxe2x80x9d may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryote or eukaryote.
With respect to RNA molecules of the invention, the term xe2x80x9cisolated nucleic acidxe2x80x9d primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a xe2x80x9csubstantially purexe2x80x9d form (the term xe2x80x9csubstantially purexe2x80x9d is defined below).
With respect to protein, the term xe2x80x9cisolated proteinxe2x80x9d or xe2x80x9cisolated and purified proteinxe2x80x9d is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in xe2x80x9csubstantially purexe2x80x9d form.
The term xe2x80x9csubstantially purexe2x80x9d refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like). With respect to antibodies of the invention, the term xe2x80x9cimmunologically specificxe2x80x9d refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., MOAT-B, MOAT-C or MOAT-D), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
With respect to nucleic acids and oligonucleotides, the term xe2x80x9cspecifically hybridizingxe2x80x9d refers to the association between two single-stranded nucleotide molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed xe2x80x9csubstantially complementaryxe2x80x9d). When used in reference to a double stranded nucleic acid, this term is intended to signify that the double stranded nucleic acid has been subjected to denaturing conditions, as is well known to those of skill in the art. In particular, the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al., 1989):
Tm=81.5xc2x0 C.+16.6 Log [Na+]+0.41(% G+C)xe2x88x920.63 (% formamide)xe2x88x92600/#bp in duplex
As an illustration of the above formula, using [Na+]=[0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57xc2x0 C. The Tm of a DNA duplex decreases by 1-1.5xc2x0 C. with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42xc2x0 C. Such sequences would be considered substantially homologous to the nucleic acid sequences of the invention.
The nucleic acids, proteins, antibodies, cell lines, methods, and kits of the present invention may be used to advantage to identify targets for the development of novel agents which inhibit the aberrant transport of cytoxic agents out of tumor cells. The transgenic mice of the invention may be used an in vivo model for chemotherapy. resistance.
The human MOAT molecules methods and kits described above may also be used as research tools and will facilitate the elucidation of the mechanism by which tumor cells acquire a drug resistant phenotype.